Missense mutations in heterogeneous ribonucleoproteins A1 and A2 (hnRNPA1D262V and hnRNPA2D290V) cause inclusion body myopathy with frontotemporal dementia, Paget's disease of bone, and amyotrophic lateral sclerosis, a complex disorder known as multisystem proteinopathy (MSP). Thus, hnRNPA1 and hnRNPA2 join the ranks of RNA-binding proteins with prion-like domains that are implicated in the pathogenesis of devastating and untreatable human neurodegenerative diseases. The mechanism by which hnRNPA1 and hnRNPA2 cause pathology in MSP patients, however, is unknown. Based on preliminary data, I hypothesize that hnRNPA1D262V and hnRNPA2D290V must misfold and bind RNA to confer cytotoxicity. I have established a yeast model that recapitulates the toxicity and mislocalization of hnRNPA1D262V and hnRNPA2D290V to cytoplasmic foci seen in human disease. I have shown that, in yeast, both hnRNPs require an intact RNA recognition motif (RRM) and some portion of the prion-like domain to confer toxicity, and that this toxicity can be suppressed by disruption of RNA-binding capability through RRM missense mutations. I will investigate the domain requirements and RNA-binding requirement for misfolding and toxicity in cultured motor neurons using a series of truncation and deletion protein constructs and RNA-binding deficient constructs. The ability to suppress hnRNPA1D262V and hnRNPA2D290V toxicity would provide a therapeutic strategy for MSP patients. Thus, I will investigate candidate toxicity suppressors that have shown promise for other neurodegeneration-linked RNA-binding proteins with prion-like domains, including TDP-43 and FUS. A potentiated variant of the yeast protein disaggregase Hsp104 appears to mitigate the toxicity of prion-like proteins via disaggregation of misfolded structures, and I have demonstrated that it has the ability to suppress the toxicity of hnRNPA2D290V but not hnRNPA1D262V. Deletion of the lariat debranching enzyme, Dbr1, ameliorates TDP-43 and FUS toxicity, and will also be explored as a strategy for suppressing the toxicity of hnRNPA1D262V and hnRNPA2D290V. In addition, a yeast deletion screen will be used to uncover additional toxicity suppressors, which will offer insight into the mechanisms underlying toxicity and reveal potential therapeutic targets. Our studies will deepen our understanding of the mechanisms underlying MSP and the link between neurodegeneration and prion-like RNA-binding proteins.